One of the major health concerns with exposure to primary vesicating agents nitrogen mustard (NM) and sulfur mustard (SM) is incapacitating skin injury with delayed vesication apart from other organ injuries. Currently, we lack effective therapies to rescue skin injuries by NM and SM in part due to a) lack of appropriate models which have the feasibility of conducting laboratory studies to evaluate promising agents'therapeutic efficacy and b) limited understanding of the mechanisms of action of NM and SM skin injury which could help develop mechanism-based therapeutics to rescue skin injuries by them. To address these two challenges, during current funding period, we established skin injury models showing that a SM analog CEES causes cell cycle arrest, inhibits DNA synthesis, and induces apoptosis in cell culture, and DNA damage, apoptosis, infiammation and micro-vesication in mouse skin. Mechanistic studies identified that CEES causes DNA damage and activation of ATM/ATR and caspases leading to cell cycle arrest and apoptosis in epidermal cells in culture. In mouse skin studies, CEES-caused oxidative stress, activation of transcription factors API and NF-KB involving MAPKs and Akt pathways, and induction of infiammatory mediators COX-2, INOS and MMP9. In mouse skin, CEES also caused H2A.X and p53 phosphorylation supporting its DNA-damaging effects, and increased myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, which could be a key player in CEES-caused oxidative stress and macromolecular damage leading to skin injuries. Employing these established models and identified mechanisms, we next performed efficacy studies with catalytic antioxidant AEOL 10150, which is the lead agent from project #2 and fiavanone silibinin, which is a dietary supplement currently in phase II and phase I clinical trials for prostate and skin cancers, respectively. Both agents strongly rescued skin injuries bv CEES. Furthermore, to develop a more relevant model in laboratory settings, we expanded our studies with primary vesicating agent NM based on its: 1) potential for being a useful bridge between completed CEES and proposed SM studies, 2) alkylating and vesicating properties, and 3) usefulness as an effective tool for the screening of new and next generation agents before moving forward with studies in another primary agent SM skin injury model. Completed studies show that NM causes: a) keratinocyte cell death in culture at much lower concentrations than CEES, and b) a strong micro-vesication and a robust neutrophil infiltration in dennis together with a five-fold higher MPO activity induction than CEES when applied topically on to the dorsal skin of SKH-1 hairiess mice. Unlike last cycle, what new current competing renewal grant offers is an on-site (in Denver) primary agent nitrogen mustard studies to: 1) fully characterize and establish NM-induced skin injury models and identify associated mechanism(s) of action, 2) develop and optimize novel therapies with AEOL 10150 and silibinin for NM skin toxicity in mice, 3) determine mechanisms of action of AEOL 10150 and silibinin therapies for NM skin toxicity, and 4) apply optimized therapies for NM to SM mouse skin injury model and confirm mechanism(s) of action. Completion of these studies will set the stage to develop identified therapies for effective rescue of skin injuries by primary vesicating agents NM and SM in an event of human exposure.